CtXR (xylose reductase from the yeast Candida tenuis; AKR2B5) can utilize NADPH or NADH as co-substrate for the reduction of D-xylose into xylitol, NADPH being preferred approx. 33-fold. X-ray structures of CtXR bound to NADP+ and NAD+ have revealed two different protein conformations capable of accommodating the presence or absence of the coenzyme 2′-phosphate group. Here we have used site-directed mutagenesis to replace interactions specific to the enzyme–NADP+ complex with the aim of engineering the co-substrate-dependent conformational switch towards improved NADH selectivity. Purified single-site mutants K274R (Lys274→Arg), K274M, K274G, S275A, N276D, R280H and the double mutant K274R–N276D were characterized by steady-state kinetic analysis of enzymic D-xylose reductions with NADH and NADPH at 25 °C (pH 7.0). The results reveal between 2- and 193-fold increases in NADH versus NADPH selectivity in the mutants, compared with the wild-type, with only modest alterations of the original NADH-linked xylose specificity and catalytic-centre activity. Catalytic reaction profile analysis demonstrated that all mutations produced parallel effects of similar magnitude on ground-state binding of coenzyme and transition state stabilization. The crystal structure of the double mutant showing the best improvement of coenzyme selectivity versus wild-type and exhibiting a 5-fold preference for NADH over NADPH was determined in a binary complex with NAD+ at 2.2 Å resolution.
A novel strictly NADPH-dependent Pichia stipitis xylose reductase constructed by site-directed mutagenesis
